Defective kernel 66 encodes a GTPase essential for kernel development in maize

Abstract The mitochondrion is a semi-autonomous organelle that provides energy for cell activities through oxidative phosphorylation. In this study, we identified a defective kernel 66 (dek66)-mutant maize with defective kernels. We characterized a candidate gene, DEK66, encoding a ribosomal assembly factor located in mitochondria and possessing GTPase activity (which belongs to the ribosome biogenesis GTPase A family). In the dek66 mutant, impairment of mitochondrial structure and function led to the accumulation of reactive oxygen species and promoted programmed cell death in endosperm cells. Furthermore, the transcript levels of most of the key genes associated with nutrient storage, mitochondrial respiratory chain complex, and mitochondrial ribosomes in the dek66 mutant were significantly altered. Collectively, the results suggest that DEK66 is essential for the development of maize kernels by affecting mitochondrial function. This study provides a reference for understanding the impact of a mitochondrial ribosomal assembly factor in maize kernel development.


Fig. S11. Polysome profiling assay with sucrose density gradient
The OD254 absorption was monitored together with fractionation.The fractions containing 40S, 60S and 80S of ribosome, and polysomes in WT and dek66 are indicated.
Table S1.List of primers used in this study.

Primer Name
Primer Sequence Application

Fig. S6 .
Fig. S6.Functional complementation test of dek66 and kernel traits of DEK66 transgenic lines (OE-1, OE-2, and OE-3).(A) Western blot assay of DEK66 protein level of transgenic lines.CK, non-transgenic control.(B) qPCR assay of DEK66 mRNA level of transgenic lines.(C) Schematic diagram of dek66 transgenic complementation.A, DEK66; a, dek66; +, transgenetransformed DEK66; -, untransformed.(D) Phenotypic analysis of transgenic kernels of the complementation test.Representative kernels with the WT (1-12) and mutant (13-24) phenotypes.Scale bars, 2 cm.(E-F) Genotype of the kernels for dek66 locus using the indel marker (Indel-1F/R).(G-H) The primers of the DEK66 gene (a forward primer named US5-1 was designed at the 3′end of maize Ubiquitin promoter and the reverse primer named DEK66-R was designed at the coding region of DEK66 gene) were used to identify the transgenic positive and negative kernels.+, expression vector plasmid as positive control.-, H2O as negative control.(I-J) CK and DEK66 transgenic lines (OE-1, OE-2, and OE-3) mature kernels randomly selected from self-pollinated ear.Scale bars, 2 cm.(K-M) Comparison of the length, width, and hundred-grain weight of randomly selected mature kernels.Error bars indicate the standard deviation (SD), ns refers to P > 0.05, Student's test).

Fig. S9 .
Fig. S9.DEK66 regulates genes involved in diverse processes.(A) Volcano plots were used to visualize RNA-seq data.Each point corresponds to a DEG.Orange and blue plots represent up-regulated and down-regulated genes, respectively.(B) Enriched GO terms in dek66 and WT endosperm differentially expressed genes.(C) Pathway functional enrichment of DEGs.X axis represents enrichment factor.Y axis represents pathway name.(D) Log2 fold change heat maps of differentially expressed genes.

Fig. S10 .
Fig. S10.Analysis of mitochondrial related differentially expressed genes.(A) Volcano plots were used to visualize RNA-seq data.Each point corresponds to a DEG.Orange and blue plots represent up-regulated and down-regulated genes, respectively.(B) Pathway functional enrichment of DEGs.X axis represents enrichment factor.Y axis represents pathway name.(C) Enriched GO terms in dek66 and WT endosperm differentially expressed genes.